Abstract
Purpose :
The majority of phototransduction proteins are peripheral membrane proteins. We know that they are localized to the photoreceptor outer segment (OS). However, their distribution within the outer segment and along the discs is poorly understood. Our recent publication using peripheral membrane probes shows that there is inhomogeneities in their distributions forming gradients along the outer segment length (Maza et al., 2019). Our preliminary results also show potential inhomogeneities along the disc surfaces as well. Our study aims to understand, mechanistically, how the inhomogeneous distributions are generated.
Methods :
Lipidated EGFP probes and probes consisting of EGFP labeled peptides from photoreceptor proteins were expressed transgenically in Xenopus rod photoreceptors. Rods in the explant retina were imaged using two-photon time-resolved fluorescence anisotropy imaging (2P-trFAIM). In this approach, a highly polarized Ti:S laser (λ=820 nm) was used to excite the EGFP labeled molecules in the photoreceptors. The polarization specific emission was recorded by two orthogonally positioned single-photon detectors that were placed after a polarizing beam splitter in the emission path. Time-resolved polarization images of the rods were gathered and analyzed for anisotropy decay-based homoFRET.
Results :
Prenylated probes formed a gradient from the base to the tip of the OS while the myristoylated probes were homogenous throughout the OS. Both probes displayed the time-dependent loss of polarization. However, the prenylated, especially farnesylated, probes contained a fast decay phase corresponding to homo-FRET. The anisotropy decay of myristoylated probes was monotonic and slow, corresponding to rotational diffusion. Similar, distribution and FRET differences were seen in the rhodopsin kinase and transducin α peptide probes.
Conclusions :
Our results demonstrate that post-translational lipidation plays a key role in OS distribution and enrichment along the disk surface. This suggests the possibility that probes are being partitioned into different membrane nanodomains based on their lipidation properties.
Maza, N. A., Schiesser, W. E., & Calvert, P. D. (2019). An intrinsic compartmentalization code for peripheral membrane proteins in photoreceptor neurons. J Cell Biol, 218(11), 3753-3772.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.